Low-Volume Sequencing System and Method of Use

ABSTRACT

Various embodiments of a low-volume sequencing system are provided herein. The system can include a low-volume flowcell having at least one reaction chamber of a defined volume (e.g., less than about 100 μl). The system can also include an automated reagent delivery mechanism configured to reversibly couple with the inlet port corresponding to a target reaction chamber thereby placing allowing for reagent to be accurately moved from a storage container to the reaction chamber with minimal reagent waste. The flowcells can include a plurality of reaction chambers (e.g., 6) thereby allowing for parallel analysis of multiple samples. Various methods of analyzing a biomolecule are also provided herein.

RELATED APPLICATIONS

This application claims priority pursuant to 35 U.S.C. §119(e) to U.S.Provisional Patent Application Ser. No. 61/238,633, filed on Aug. 31,2009, entitled “Enhanced System and Methods For Sequence Detection,”U.S. Provisional Patent Application Ser. No. 61/238,667, filed on Aug.31, 2009, entitled “Enhanced Flow Cell and Reagent Delivery For SequenceDetection,” U.S. Provisional Patent Application Ser. No. 61/307,623,filed on Feb. 24, 2010, entitled “Methods of Bead Manipulation andForming Bead Arrays,” U.S. Provisional Patent Application Ser. No.61/307,492, filed on Feb. 24, 2010, entitled “Flowcells and Methods ofFilling and Using Same,” U.S. Provisional Patent Application Ser. No.61/307,641, filed on Feb. 24, 2010, entitled “Flowcells and Methods ofFilling and Using Same,” and U.S. Provisional Patent Application Ser.No. 61/307,486, filed on Feb. 24, 2010, entitled “Flowcell, FlowcellDelivery System, Reagent Delivery System, and Method For SequenceDetection,” the entirety of each of these applications beingincorporated herein by reference thereto.

FIELD

The present disclosure is directed towards molecular sequencing, inparticular towards low-volume flowcell design and reagent deliveryoptimization.

BACKGROUND

Nucleic acid sequencing techniques are of major importance in a widevariety of fields ranging from basic research to clinical diagnosis. Theresults available from such technologies can include information ofvarying degrees of specificity. For example, useful information canconsist of determining whether a particular polynucleotide differs insequence from a reference polynucleotide, confirming the presence of aparticular polynucleotide sequence in a sample, determining partialsequence information such as the identity of one or more nucleotideswithin a polynucleotide, determining the identity and order ofnucleotides within a polynucleotide, etc.

Next generation sequencing techniques commonly utilize fluidictechnologies for performing aspects of sample analysis. For example,Assignee's PCT Application Publication No. WO 2006/084132, entitled“Reagents, Methods, And Libraries for Bead-Based Sequencing,” theentirety of which is incorporated herein by reference thereto, providesvarious techniques, systems, and methods for sequencing a sample coupledto a solid-support (e.g., a bead, particle, surfaces and surfacefeatures, etc.) wherein a plurality of supports are disposed over thesurface of a flowcell. Flowcells allow for a large number of samples, orsamples coupled to other solid-supports, to be immobilized in randomand/or ordered fashion across reaction chamber(s) while reagents areadded to, removed from, or pumped through the chamber(s) to produce thedesired effect (e.g., reaction, wash, etc.). Typical systems can alsoinclude imaging, optics, or other detection components in communicationwith the reaction chambers thereby allowing sample images or otherproperties to be rapidly captured and analyzed.

In view of the ever-increasing benefits of genomic analysis, demandcontinues for, among other things, faster sample analysis, higherthroughput, enhanced sequence accuracy, and reduced cost (e.g., on aper-run or per-genome basis).

SUMMARY

Various embodiments of a sample analysis and sequencing system areprovided herein. In some embodiments, the system includes a low-volumeflowcell having at least one reaction chamber of a defined volume withat least one fluid transfer port (e.g., inlet/outlet port). The systemscan also include an automated reagent delivery mechanism having afluidic dispenser with an internal reservoir such as a compartmentconfigured to retain an amount of reagent. In some exemplaryembodiments, the dispenser can include a distal portion configured toreversibly couple with the inlet port thereby placing an internalcompartment of the dispenser into fluid communication with at least onereaction chamber, and optionally further configured to dispense to thereaction chamber a volume substantially equal to the defined volume ofthe reaction chamber.

The systems can also include a controller (e.g., a computer, processor,etc.) in communication with the delivery mechanism, and configured tocouple/decouple the delivery mechanism with the inlet port, and furtherconfigured to dispense reagent to the reaction chamber. In someembodiments, instructions for coupling and/or decoupling the deliverymechanism are embodied in hardware, firmware, software, or combinationsthereof. In some aspects, the instructions can be contained incomputer-readable media and optionally transferable between differentsystems.

The delivery mechanism can be configured in some embodiments to withdrawa volume of reagent substantially equal to the volume of the reactionchamber from a storage container and transfer this volume of reagent tothe reaction chamber. Further, volume of the reaction chamber can be ina range from as low as flowcell manufacturing technology will allow andabout 100 between about 10 μl and about 40 μl, between about 20 μl andabout about 25 μl, etc. These features can, among other things, assistin reducing reagent use and in reducing or eliminating reagent waste.

Flowcells of the systems can include a single reaction chamber of adiscrete volume or can include a plurality of reaction chambers eachhaving a discrete volume. The volume of each reaction chamber can be thesame for each chamber or different. The reaction chamber(s) can extendbetween distinct inlet and outlet ports, or can have a common inlet andoutlet ports. In those embodiments having a plurality of reactionchambers, the flowcells can have 2, 3, 4, 5, 6, 7, 8, etc. chambers.Additionally, when delivering reagent to multiple chambers, one or moreinternal compartments of the delivery mechanism can be configured totransfer/deliver a volume of a reagent substantially equal to a totalvolume of the plurality of reaction chambers.

Various flowcell configurations are also disclosed herein. For example,at least one fluid transfer port can be incorporated into a top portionof the flowcell, or can be incorporated into a bottom portion of theflowcell. Also, at least a portion of the flowcell's surface can beconfigured to bind, immobilize, or trap a sample or other reagents orcomponents (e.g., polynucleotides, solid-supports configured to bind apolynucleotide). In some embodiments, the flowcells can include aninterior portion configured to bind, immobilize, or trap sample,reagents, or other components.

In some embodiments, the reagent delivery mechanism includes a roboticassembly having x-, y-, and z- functionality. Additionally, the systemcan include a reagent storage container housing a plurality of reagents(which can be in the form of a kit). The delivery mechanism can beconfigured to retrieve sample from a storage container and dispensereagent into a reaction chamber without plumbing or tubing requirementstherebetween. In some exemplary embodiments, dead volume associated withplumbing, tubing, or other fluid connection means can be reduced oreliminated, which can assist in reducing reagent use, reducing oreliminating reagent waste, and enhancing reagent delivery and systemaccuracy and efficiency.

Various embodiments of a low-volume flowcell are also provided herein.In some embodiments, the flowcell includes a substrate having at leastone reaction chamber of a defined volume extending between an inlet portand an outlet port, the inlet port being configured to reversibly couplewith an automated reagent delivery mechanism which is configured todeliver a pre-determined amount of reagent to the reaction chamber. Toreduce reagent requirements and waste, the reaction chamber can be sizedand configured to minimize the pre-determined amount of reagent requiredto affect a desired result. For example, the defined volume of thereaction chamber can be a volume within a range of between about aminimum amount (e.g., to limits of flowcell manufacturing technology)and about 100 μl between about 10 μl and about 40 μl or about 25 μl.

Methods of analyzing biomolecules are also provided herein, includingmethods employing various embodiments of flowcells and reagent deliverymechanisms disclosed herein. In some embodiments, a method can includeproviding an automated reagent delivery mechanism configured to withdrawa pre-determined volume of a reagent from a reagent storage container,withdrawing a pre-determined amount of the reagent from the storagecontainer by the automated reagent delivery mechanism, and coupling aportion of the automated reagent delivery mechanism to an inlet port ofa flowcell, the inlet port being in fluid communication with a reactionchamber having a defined volume. Methods can also include dispensinginto the reagent chamber a reagent volume substantially equal to thedefined volume of the reagent chamber, and decoupling the automatedreagent delivery mechanism from the inlet port.

In those embodiments relating to flowcells with multiple chambers (e.g.,2, 3, 4, 5, 6, 7, 8, etc.), methods of analyzing biomolecules caninclude repeating the coupling and decoupling steps for each of thecorresponding inlet ports in fluid communication with distinct reactionchambers, each reaction chambers having discrete volumes which can bethe same or different than other reaction chambers. In one embodiment,the method can include dispensing the same reagent to each (or at leasttwo) of the plurality of chambers, and other embodiments can includedelivering different reagents to each (or at least two) of the chambers.

Instructions for performing or implementing the various functions andfeatures described above and in the remainder of this disclosure can beembodied in hardware, firmware, software, or combinations thereof.Instructions can also be contained in computer-readable media, andoptionally be transferable between different systems and reconfigurableand updatable via on-line services, by the user, or by other processesand means.

These and other embodiments are described in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will be more fully understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 is an overview of a preferred embodiment of the presentlydisclosed low-volume sample analysis and sequencing system;

FIG. 2 is another representation of the low-volume sequencing system ofFIG. 1;

FIG. 3 is a representation of a preferred embodiment of a sequencingapparatus of the present disclosure;

FIG. 4 is another representation of the sequencing apparatus of FIG. 3;

FIG. 5A is an exploded view of a preferred embodiment of the presentlydisclosed low-volume flowcell;

FIG. 5B is a top view of the low-volume flowcell of FIG. 5A;

FIG. 6A is an exploded view of another preferred embodiment of thepresently disclosed low-volume flowcell;

FIG. 6B is a top view of the low-volume flowcell of FIG. 6A;

FIG. 7 is a cross-sectional view of an alternative embodiment of thepresently disclosed low-volume flowcell;

FIG. 8 is a representation of a plurality of imaging panels associatedwith a reaction chamber of the presently disclosed flowcell;

FIG. 9 is a side-view of an embodiment of a clamping mechanismconfigured to secure a flowcell relative to a processing stage;

FIG. 10A is a top view of an embodiment of a flowcell processing stageand associated clamping mechanism;

FIG. 10B is a side-view of an embodiment of a flowcell clamped to theprocessing stage by the clamping mechanism;

FIG. 11A is a representation of an embodiment of a flowcell in relationto a thermal block;

FIG. 11B is a graph showing substrate deflection under uniform windowloading;

FIG. 11C is a graph showing deflection of a glass window under 1 psiload;

FIG. 11D is a graph showing deflection of a sapphire window under 1 psiload;

FIG. 12 is representation of another embodiment of the presentlydisclosed flowcell;

FIG. 13 is a representation of a flowcell relative to an objective lens;

FIG. 14 is a representation of another embodiment of the presentlydisclosed flowcell;

FIG. 15 is a representation of another embodiment of the presentlydisclosed flowcell;

FIG. 16A is a view of a preferred embodiment of a presently discloseddeposition tool positioned relative to a low-volume flowcell;

FIG. 16B is a view of the deposition tool of FIG. 16A coupled to theflowcell thereby forming a deposition assembly;

FIG. 17 is a representation of a preferred embodiment of a precisionreagent delivery mechanism;

FIG. 18 is a representation of an embodiment of the presently disclosedreagent coupling mechanism;

FIG. 19 is another representation of the preferred embodiment of theprecision delivery mechanism;

FIG. 20 is a schematic diagram of a preferred embodiment of a presentlydisclosed computer system; and

FIG. 21 is a schematic diagram of an embodiment of a system forprocessing a sample.

DETAILED DESCRIPTION

Low-volume sample analysis or sequencing systems and methods of use areprovided herein. The presently disclosed system utilizes variousembodiments of a low-volume analysis apparatus and a precision reagentdelivery mechanism. The system can include an embodiment of a low-volumeflowcell having discrete reaction chambers sized and configured tominimize reagent volume necessary to achieve the desired result (e.g.,reaction, wash, etc.). For example, various embodiments reduce reagentvolume per chamber to within a range of between about a minimal amountpossible in view of flowcell manufacturing technology and about 100 μl.The precision reagent delivery mechanism can include an x-y-z functionalrobot capable of receiving a pre-determined amount of reagent from areagent storage container, reversibly coupling to a dedicated fluidtransfer port (e.g., an inlet port), and dispensing this minimal volumeto the reaction chamber. In one embodiment, the pre-determined amount ofreagent is substantially equal to the amount of reagent to be dispensedinto the reaction chamber thereby substantially eliminating any amountof reagent waste. As detailed below, such a system significantly reducesreagent requirements thereby providing cost savings and simplifyingmaintenance over typical systems utilizing extensive plumbing/tubingrequirements.

The analysis systems, flowcells, fluid and reagent delivery systems,software, and other systems, apparatuses, and methods disclosed hereincan be used in connection with various sequencing techniques andprocesses, such as chain termination or dideoxynucleotide sequencing,chemical degradation sequencing, sequencing by synthesis,pyrosequencing, sequencing by hybridization, oligonucleotide-basedsequencing, and single-molecule sequencing. The analysis systems,flowcells, fluid and reagent delivery systems, software, and othersystems, apparatuses, and methods disclosed herein can also be used inconnection with automated, partially automated, and manual sequencinginstruments and processes. For example, low-volume flowcells disclosedherein can be used for performing next-generation sequencing reactionssuch as oligonucleotide-based reactions and sequencing-by-synthesisreactions. An advantage of some embodiments of the disclosed flowcellsis a reduction in the amount of sample or reagent that is used; wheresuch samples or reagents are available only in small quantities or areexpensive or otherwise difficult to make or acquire, substantialreduction in cost can be realized (sequencing of a whole genome for$1,000 or less) and the sequencing of samples that were previouslydifficult or impossible to sequence.

Certain exemplary embodiments will now be described to provide anoverall understanding of the principles of the structure, function,manufacture, and use of the systems and methods disclosed herein. One ormore examples of these embodiments are illustrated in the accompanyingdrawings. Those skilled in the art will understand that the systems andmethods specifically described herein and illustrated in theaccompanying drawings are non-limiting exemplary embodiments and thatthe scope of the present disclosure is defined solely by the claims. Thefeatures illustrated or described in connection with one exemplaryembodiment may be combined with the features of other embodiments. Suchmodifications and variations are intended to be included within thescope of the present disclosure.

FIG. 1 provides an overview of a preferred embodiment of the presentlydisclosed low-volume sample analysis and sequencing system 10. As shown,the system 10 includes a low-volume analysis apparatus 12 incommunication with a precision fluid delivery mechanism 26.Additionally, the system 10 includes a controller 30 (e.g., acomputer/processor running control software) in communication with anyor all components of the analysis apparatus 12 and/or the precisiondelivery mechanism 26. As detailed below, the controller 30 can directvarious aspects of the apparatus 12 and/or delivery mechanism 26,implement various steps, control reaction conditions, acquire data,store data, perform data analysis, communicate with additional automatedmodules, etc. The controller 30 can also be in communication with a userinterface 32 configured to allow a user to, for example, input sampleinformation, select desired reaction conditions, monitoranalysis/reaction progression, review analysis results, provide processcontrol, etc.

While the apparatus 12 is shown to include a variety of components,those skilled in the art will appreciate that any or all of thesecomponents may be removed, substituted, new components added, etc.Additionally, any or all of these components may be considered to beindependent of the apparatus 12. All such variations are within thespirit and scope of the present disclosure.

In the preferred embodiment, the apparatus 12 includes a low-volumeflowcell 14 in communication with a temperature block 16 and processingstage 18. As detailed below, the low-volume flowcell 14 can beconfigured to retain sample in any number of discrete reactionschambers. The reaction chambers can likewise be sized and configured tominimize reagent volume required to affect the desired result withineach chamber. That is, the width, length, and/or height of the chambercan be selected to maintain desired flow characteristics while ensuringthe desired reaction takes place. Additionally, the discrete chambersallow for multiple, distinct reactions or distinct samples to beanalyzed simultaneously, or the same reaction may be performed multipletimes in parallel as a quality control check. Thus, the entire surfacearea of the flowcell 14 does not need to be utilized when a singlechamber (e.g., 1 of 6 available chambers) can provide the desired resultthereby limiting the required reagent volume. Additionally, the system10 can be configured to include multiple independently operatedflowcells (e.g., 2 flowcells located on the processing stage 18). Invarious embodiments, the low volume characteristics of the flowcell maybe determined on the basis of the amount of reagents needed toeffectuate an efficient reaction between the sample and the reagentswith little or no excess. For example, if the principle reactivitybetween the sample and the reagents takes place only in close proximitywith the flowcell surface with little or no reaction taking place in thevolume contained above the flowcell surface then the cross-sectionalarea or overall volume of the flowcell may be decreased such thatreagents contained within the flowcell are principally positioned wherereactivity is substantial or optimal.

In maintaining an accurate temperature profile within each reactionchamber, a temperature block 16 can be in placed into thermalcommunication with the flowcell(s). Various such temperature blocks arewithin the spirit and scope of the present disclosure. For example, thetemperature block 16 can be a device capable of both heating andcooling, such as a peltier device. Other temperature control devices canalso be used, such as heating/cooling units or blocks, thermalcontrollers, heat transfer devices, reaction processors. In someembodiments, there is a water-based variant referred to as a hydrocyclerwhich uses as temperature controlled water bath. In some embodiments,thermocycling of the reaction components (e.g., cycling between higherand lower temperatures) is not necessarily required and the reaction mayalso proceed isothermally which may or may not require a heat block(e.g., reaction proceeds at ambient temperature). As detailed below,flowcell temperature control is facilitated by the reduced reagentvolume requirements of the presently disclosed system where lowerfluidic volumes may be thermally regulated more efficiently and quickly(e.g., lower heat capacity in the fluidic volume allowing faster rampingin heating and cooling) because the system no longer must account forlarge fluctuations in reagent volume.

The flowcell 14 and temperature block 16 can be coupled to a movable ormotorized processing stage 18 configured to move between, for example, aflowcell loading stage position and an analysis position. Those skilledin the art will appreciate that various types of motors can be utilizedto drive the processing stage 18 between such positions.

In the analysis position, the low-volume flowcell and associated sampledisposed therein can be placed into communication with an optics/imagingmechanism 20. The optics/imaging mechanism 20 may be configured totransmit and/or filter excitation energy from an excitation source(e.g., an arc lamp, a laser, etc.) through one or more lenses, filters,etc. such that a sample (or selected area or panel of the flowcell) isimaged at one or more selected wavelengths prior to moving onto the nextsample (or selected area or panel of the flowcell). The optics/imagingmechanism 20 can further be configured to include one or more filters(not shown) such that the emission signals pass through a correspondingfilter prior to impinging upon the detector 24 (e.g., a CCD). Oneembodiment of the optics/imaging mechanism 20 is described in greaterdetail in co-pending U.S. patent application Ser. No. ______, filed Aug.31, 2010, entitled “Fast-Indexing Filter Wheel and Method of Use,” theentirety of which is incorporated herein by reference thereto.

The fluid delivery mechanism 26 can include an x-y-z robot configured toaccurately receive a predetermined amount of reagent from a reagentstorage container 28, reversibly couple to a fluid transfer port (e.g.,an inlet port) in fluid communication with a target reaction chamber,and dispense the selected volume of reagent to the chamber. In oneembodiment, the fluid delivery mechanism is configured to eliminate orat least substantially reduce the amount of reagent utilized in typicalsystems by eliminating commonly found extensive plumbing/tubingrequirements. Additionally, the mechanism 26 can be configured to retainand deliver a volume of reagent that is substantially identical to thevolume of the reaction chamber thus essentially eliminating any wastedreagent. The mechanism 26 can also include various sensors capable ofdetermining when a reagent reservoir needs to be filled, capable ofaccurately determining volume within the reservoir, etc. In variousembodiments, the method of fluidic dispensation described hereindesirably reduces potential wasted reagents that would be contained inthe instrument plumbing and fluidic transfer lines. Furthermore, thesystem desirably improves thermal control over the sample and/orreagents which may be held at desired temperatures and dispensed asdesired without substantial temperature deviations which may accompanymovement through complex instrument plumbing and fluid transfer lines.

FIGS. 2-4 provide various representations of an embodiment of thepresently disclosed sequencing system 10 and apparatus 12. That is, FIG.2 shows an embodiment of an instrument shell 11 housing the systemapparatus 12 and the precision fluid delivery mechanism 26. As shown,the apparatus 12 can include a detector 24 (e.g., a CCD) disposed alonga path in optical communication with the flowcell 14 with the reagentdelivery mechanism 26 and reagent storage container 28 positioned inproximity to the apparatus 12. Various sizes and configurations of theinstrument shell 11 are within the spirit and scope of the presentdisclosure. That is, the shell 11, apparatus 12, and reagent deliverymechanism 26 can be sized and configured to be positioned on a labbench. Those skilled in the art will appreciate that any scale of thesystem 10 is within the spirit and scope of the present disclosure.

FIG. 3 is another representation of the apparatus 12 showing variouscomponents coupled to a frame 34. The frame 34 can provide stabilitywhile maintaining a desired relationship between components. Forexample, the detector 24 and optics/imaging mechanism 20 can be coupledto the frame 34 such that flowcell can be reliably moved into/out ofcommunication with these components.

FIG. 4 provides another representation of the apparatus 12 with variouscomponents removed for clarity. The processing stage 18 can be coupledto a motor (not shown) such that the stage 18 can be moved between theloading position (as shown) and analysis position. Furthermore, themotor can be used to timely control positioning of the stage withrespect to the optics such that a selected portion of the flowcell canbe imaged and wherein the flowcell can be repositioned to anotherselected position via stage movement to image another portion of thesample contained within the flowcell. FIG. 4 also provides an embodimentof the relationship between the excitation source 22, the optics/imagingmechanism 20, and detector 24. As shown, the excitation source 22 andoptics/imaging mechanism 20 can be positioned above the flowcell 14. Inother embodiments, the source 22 and mechanism 20 can be positionedbelow the flowcell 14.

FIG. 5A provides an exploded view of a preferred embodiment of thepresently disclosed low-volume flowcell 14. As shown, the flowcell 14can include a top-layer 36, a middle-component 38, and a bottom-layer40. The top-layer 36 can include one or more inlet ports 44 and/oroutlet ports 48 in fluid communication with a plurality of reactionchannels/chambers 42 extending along a length of the flowcell 14. Theflowcell 14 can also include a middle-layer defining the reactionchannels/chambers 42, and a bottom-layer 40. In other embodiments, theflowcell 42 can include any alternative number of layers. That is, theflowcell can comprise a single substrate positioned relative to anothercomponent of the apparatus (e.g., a heat block), two layers wherein thetop and/or bottom layers are sized and configured to define the reactionchambers 42, etc. As such, those skilled in the art will appreciate thatany such configuration of flowcell capable of defining at least onereaction chamber 42 is within the spirit and scope of the presentdisclosure.

In use, sample can be incorporated/bound to either or both the top andbottom surfaces of the flowcell. The target sample can be bound directlyon the substrate layer or can be coupled to solid-supports (e.g.,particles or beads) which can then bind to the desired substrate. Invarious embodiments, the target sample can be bound to both the top andbottom layers to achieve a higher target sample density and furthermoreto achieve more efficient utilization of reagents in the flowcell. In apreferred embodiment, sample is bound to solid-supports which are thenbound to the top layer of the flowcell 14.

The inlet port 44 of the flowcell 14 allows for, as detailed below, theprecision reagent delivery mechanism 26, to deliver a substantiallyexact pre-determined amount of reagent to the target reaction chamber(s)42. Additionally, the outlet port 46 allows for waste to be removed fromthe chamber(s) 42. While each chamber 42 is shown to be in communicationwith a distinct outlet port 46, other embodiments can include a singleoutlet port in communication with all chambers. In such an embodiment,waste streams from each chamber 42 can pool together prior to exitingthe flowcell from the one outlet port. In one embodiment, the fluidicsinterface (i.e., the inlet and outlet ports 44, 46) is incorporated intothe top layer 36 of the low-volume flowcell. In other embodiments, thefluidics interface can be incorporated into the bottom of the flowcell14. In another embodiment, the inlet/outlet ports can be incorporatedinto a side portion of the flowcell. In various embodiments, fluidicdispensation and withdrawal of fluid from the sample chamber can beachieved by positive or negative pressure or fluidic displacementmethods such as purging air through the sample dispenser and/orflowcell.

The presently disclosed low-volume flowcell 14 can be configured toinclude any number of discrete channels defining reaction chambers 42.Thus, the flowcell 14 can simultaneously analyze a plurality of samples,can analysis the same sample multiple times, and/or can analyze the samesample under distinct reaction conditions. Additionally, each chamber 42can be sized and configured to minimize reagent volume. That is, theheight, width, and/or length can be modified in order to reduce reactionvolume while also maintaining fluid flow characteristics (e.g., avoidingbubble formation) and also allowing for the desired reaction to occur.

As shown, the low-volume flowcell 14 can include 4 reaction chambers 42.Various other embodiments of the low-volume flowcell 14 can include anynumber of reaction chambers 42. For example, the flowcell 14 can include1, 2, 3, 4 (as shown in FIGS. 5A-5B), 5, 6 (as shown in FIGS. 6A-6B), 7,8, etc. reaction chambers 42.

FIG. 5B provides a top view of a 4-chamber low-volume flowcell 14.Various dimensions of the flowcell 14 and/or chambers 42 are within thespirit and scope of the present disclosure. For example, as shown,dimensions “A”, “B”, “C”, and “D” can be defined for the flowcell 14.The length of the flowcell 14 is shown as “A”. Flowcells 14 of variouslengths are within the spirit and scope of the present disclosure. Forexample, the length can fall within a range of about 80 mm to about 120mm, about 90 mm to about 110 mm, about 100 mm, etc. In a preferredembodiment, the length is about 95 mm. The width of the flowcell 14 isshown as “B”. Flowcells 14 of various widths are within the spirit andscope of the present disclosure. For example, the width can fall withina range of about 20 mm to about 40 mm, about 25 mm to about 35 mm, about30 mm, etc. In a preferred embodiment, the width is about 40 mm.

The length and width of an embodiment of reaction chambers of the4-chamber flowcell is represented as “C” and “D” in FIG. 5B. Varioussuch lengths are within the spirit and scope of the present disclosure.For example, the length can fall within a range of about 60 mm to about90 mm, about 70 mm to about 80 mm, etc. In a preferred embodiment, thelength is about 75 mm. Various such widths are also within the spiritand scope of the present disclosure. For example, the width can fallwithin a range of about 2 mm to about 6 mm, about 3 mm to about 5 mm,etc. In a preferred embodiment, the reaction chamber width is about 5mm.

As indicated above, to reduce reagent volume and associated waste, thechambers 42 can be sized and configured to utilize a minimum volume ofreagent while maintaining desired flow characteristics and also allowingfor the desired reaction to occur. In one embodiment, the height of thereaction chamber 42 can be configured to provide for such reagentoptimization. For example, referring again to the embodiment of the4-chamber flowcell of FIG. 5B, the height of the reaction chamber 42 canbe, for example, about 30 μm, about 50 μm, etc. In an embodiment wherethe reaction chamber height is about 30 μm, the internal volume of thechamber corresponds to about 10 μl, and in an embodiment where thereaction chamber height is about 50 μm, the internal volume of thechamber corresponds to about 17 μl. In other embodiments, each reactionchamber of a multi-reaction chamber flowcell can have a volume fallingwithin a range of between about 1 μl and about 100 μl, between about 10μl and 40 μl, between about 20 μl and about 30 μl, etc. In oneembodiment, the volume of each reaction chamber of the flowcell can beabout 25 μl. As will be appreciated by those skilled in the art,flowcells of any number of chambers having various dimensions are withinthe spirit and scope of the present disclosure.

FIGS. 6A-6B provide another embodiment of a flowcell having 6 reactionchambers 42′. Similar to above, various dimensions of the flowcell 14and/or chambers 42′ are included within the spirit and scope of thepresent disclosure. For example, as shown in FIG. 6A, dimensions “A′”,“B′”, “C′”, and “D′” can be defined for the flowcell 14. In a preferredembodiment, the length of the flowcell 14, represented by A′, can bebetween about 100 mm and about 150 mm, preferably about 128 mm. Thewidth of the flowcell 14, shown as B′, can be between about 40 mm and 80mm, preferably about 60 mm.

The length and width of an embodiment of reaction chambers of the6-chamber flowcell is represented as “C′” and “D′” in FIG. 6B. Varioussuch lengths are within the spirit and scope of the present disclosure.In one embodiment, the length can be between about 60 mm and 120 mm,preferably about 90 mm. Various such widths are also within the spiritand scope of the present disclosure. In one embodiment, the reactionchamber width can be between about 3 mm and 7 mm, preferably about 5 mm.

FIG. 7 provides a cross-sectional view of an embodiment of thelow-volume flowcell of FIG. 5B taken along E-E. As shown, the reactionchambers 42 can be configured in various manners to optimize samplebinding and/or incorporation. For example, component(s) of the flowcell42 can be configured to provide for ordered positioning of sampleswithin the reaction chamber 42. That is, as shown in FIG. 7, a portionof the flowcell 14 in communication with the reaction chamber 42 caninclude grooved elements 48 thereby allowing for sample to bedistributed within the grooved element 48. In such an embodiment,positioning sample (e.g., beads having sample coupled thereto) withinknown locations can facilitate sample identification, sample imaging,etc. A more detailed description of such ordered-array embodiments isprovided in Assignee's co-pending U.S. patent application Ser. No.______, filed August 31, 2010, entitled “Methods of Bead Manipulationand Forming Bead Arrays,” the entirety of which is incorporated hereinby reference thereto.

During imaging and analysis, defined portions of each reaction chamber42 can be imaged individually. In one embodiment, a single area isimaged at multiple wavelengths (e.g., 4 images taken of each area at 4distinct wavelengths) prior to imaging another area. FIG. 8 provides areaction chamber 42 segregated into a plurality of imaging panels 50wherein each panel 50 is imaged as discussed above. Those skilled in theart will appreciate that a reaction chamber 42 having various numbers ofimaging panels 50 are within the spirit and scope of the presentdisclosure. For example, each reaction chamber 42 can include a numberof panels 50 within a range of between about 400 imaging panels 50 toabout 1000 imaging panels 50. In a preferred embodiment, the reactionchamber 42 includes about 670 imaging panels 50. Additionally, eachimaging panel 50 can include a desired amount/density of depositedsample (e.g., beads). For example, each panel 50 can include about100,000 deposited beads, about 200,000 deposited beads, about 300,000,about 400,000, etc. In a preferred embodiment, each imaging panel 50includes about 300,000 deposited beads.

In one embodiment, the flowcell 14 is a presealed, low volume device,capable of being placed directly onto a precision monitoring orretaining surface on the stage 18, which is pre-aligned with the opticalaxis. As the flowcell 14 is pre-sealed, clamping forces, and thusdeformation forces, are very minimal. Such an arrangement can be usedwith the optics either above or below the flowcell 14. Reagentconsumption can be limited to the absolute minimum required to fill thespecially designed low-volume flowcell, thus achieving minimaloperational costs. In view of the minimal volume requirements, opticalalignment and distortion reduction is also greatly improved.

As shown in FIGS. 9, 10A, and 10B, the flowcell 14 can be mechanicallycoupled to the stage 18 via first and second clamping mechanisms 52, 54.The clamping mechanisms 52, 54 can include, for example, rotatablefingers configured to rotate into and out of communication with oppositeends of the low-volume flowcell 14. Once secured to the processing stage18, the flowcell 14 can be moved between the loading position and theanalysis position. As shown in FIG. 10B, while in the analysis position,an objective lens 56 can placed into communication with thesample/flowcell.

The various components/substrates of the flowcell can be constructed ofvarious materials and/or mixtures of materials. For example, thecomponents can include glass, sapphire, plastic, ceramic, metal, etc.Additionally, any or all of the components/substrates can be layeredcomponents. For example, one layer can include a composition capable offacilitating sample binding via a covalent coupling between the particleand substrate or between the sample and substrate. The layer may alsocomprise a surface treatment such as a plasma-oxygen treatment whichfacilitates binding of the desired sample and/or particle. Depending onthe nature of the sample (e.g., protein, lipid, nucleic acid, etc.) thelayer can be selected to provide a desired selective binding property.Similarly, particles or beads can be secured to the surface via afunctional group present on either the particle/bead or the surface, orboth.

In one embodiment, various components of the flowcell 14 can beconstructed of a moldable polymer, copolymer, and/or polymer blend thathave relatively low background fluorescence when exposed to excitationlight. Furthermore, it may be desirable for such material to exhibitlittle or no reactivity with the reagents (e.g., diminishing theactivity of any enzymes present in the reagents). Various thermoplasticsare thus suitable for use in flowcell construction including, by way ofexample cycloolefin polymers, polypropylene, polystyrene, etc. Thisresults in high quality components at relatively low cost, with lowfluorescence optical properties that further enable the incorporation ofthe molded microfeatures (e.g., positioning element 48). In variousembodiments, small particles or beads can be immobilized to thetop/bottom of the flowcell 14 by means of attachment to a functionalizedsurface. To facilitate this approach, the surface can be functionalizedprior to assembly, and the opposing surface can remain unfunctionalized.In some embodiments, functionalizing can be done after assembly, tofunctionalize both surfaces. According to some embodiments, the surfacefunctionalization can comprise the deposition of a thin layer of abinding agent such as a metal oxide that is only a few atoms thick, suchas ten or fewer or five or fewer atoms thick.

In various embodiments the flowcell can be assembled in differentmanners. Two such methods are described for the final assembly. In thefirst method, the cover surface (unfunctionalized) is bonded to the beaddeposition surface (functionalized), prior to bead deposition. The beadsare then introduced through a port in the closed assembly fordeposition. In the second method, the beads are deposited on thefunctionalized surface prior to the assembly of the opposing surfaces.For this method, a very thin (for example a few microns thick) layer ofpressure sensitive adhesive, patterned to match the channel ribs, isprovided on the mating surface.

Other flowcell configurations capable of improving system performanceare also provided herein. For example, as shown in FIG. 11A, sapphirecan be used as a substrate for deposition of sample (e.g., beads). Morespecifically, sample 58 can be disposed along the sapphire substrate 62while an objective lens 56 can image the sample through the substrate62. FIG. 11A also shows the relationship between the reaction chamber 42and the thermal block 16 while a gasket mechanism 60 defines thereaction chamber 42. Sapphire can provide various advantages over theuse of glass. For example, a glass substrate is typically about 1 mmthick with a Young's Modulus of about 64 Gpa and a bending modulus ofabout 25 MPa. In comparison, a sapphire substrate is about 5.4 timesstiffer and exhibits about 14 times the bending strength.

FIG. 11B further reveals certain desirable characteristics of sapphirein view of displacement versus load and maximum allowable stresses data.That is, the graph reflects that the maximum pressure for a glass windowsize of about 2.54×0.84 sq. inches is about 1.6 psi. For sapphire, thatvalue increases to 22 psi. FIG. 11C provides a plot of glass substratedeflection versus substrate thickness. As shown, under a load ofapproximately 1 psi, deflection can be observed to be on the order ofapproximately 0.6 microns. As shown in FIG. 11D, the equivalentstiffness and deflection of sapphire under the same conditions can beachieved with only about half the thickness. The aforementionedcharacteristics provide a mechanism by which to reduce the workingdistance with similar or better performance in deflection and betterrobustness since the sapphire substrate material is more tolerate ofgreater stress.

In another embodiment, as shown in FIG. 12, sapphire or other selectedmaterials can be used in the fabrication of a pressure chamber flowcellfor analysis of sequencing reactions. Desirably, such a flowcell canutilize a more durable or permanent optical window with robustengineering properties (such as that formed from sapphire) than can berelatively thin and permit visualization of the target of interest (forexample deposited beads).

Such a system can make use of a flexible bead substrate consumable 64(e.g., a polymer or plastic slide). The reaction chamber 42 can bepressurized facilitating the flattening of the flexible bead substrate64 against the thermal block 16 both for thermal transfer aspects aswell as for flatness of the flexible substrate 64, which is desirablefor narrow depth of field systems. The substrate/optical window 62 canbe desirably configured to be robust enough to tolerate working loads,and as described above and shown in FIGS. 11B-11D, sapphire candemonstrate pressures in the tens of psi.

Another benefit of a sapphire substrate is depicted in FIG. 13. That is,sapphire can be directly bonded to a corresponding metal frame in orderto avoid collision with an objective lens 56. That is, in certainflowcell designs, as the objective scans to view the entire slide(up/down direction in view), it can hit the carrier brackets (raisedside portions). Beneficially, an improved flowcell design can beachieved, and for which sapphire can be a desirable material, where thewindow permits direct bonding to the flowcell walls. Such a designallows for a generally flat topology that can be configured to be lowerprofile and less obstructive to the objective.

In yet another embodiment, as represented in FIG. 14, a flowcell designcan include an indium tin oxide-based (“ITO”) material 66 or similarsuch composition configured to provide transparent conductive coatingsthat can serve a multiplicity of purposes. For example, the coating 66can be used as a heater element to provide faster thermal responsetimes. Furthermore, ITO can be used as an electro-wetting electrode forfilling of a fluidic chamber with reagents thereby enabling a process tofill a flowcell that minimizes bubbles in the chamber.

In summary, these low-volume flowcell designs provide numerous potentialadvantages, including the ability to use materials with higher stiffnessand strength that enables thinner substrates, high thermal conductivitythat provides better thermal uniformity properties, optical gradequalities such as flatness and smoothness, novel fluidic fillingpossibilities to minimize bubbles, and pressurization capable offlattening the consumable for reducing focal variation.

As shown in FIG. 15, the flowcell 14 can include a plastic wall 68disposed between the reaction chamber 42 and the temperature block 16thereby requiring the system 10 to be configured to account forpotential thermal lag and/or thermal non-uniformity within the reactionchamber 42 due to heating/cooling through the relatively poor conductiveplastic wall 68. The thermal profile can be based on an internaltemperature sensor (not shown) imbedded in the center of the temperatureblock 16. In one embodiment, the system can be configured to reduce suchthermal lag by over-shooting the target temperature. Over-shooting thetarget temperature can yield a faster heat transfer to the sample andreduce thermal lag. In use, the block 16 temperature should be adjustedand tuned to account for sample temperature.

Prior to securing the low-volume flowcell to the processing stage 18 foranalysis, sample can be introduced to the reaction chambers 42. In oneembodiment, the sample is coupled to a solid-support (e.g., beads).Beads can be introduced to the chambers 42 in many manners, for example,through the inlet and/or outlet ports 44, 46. As the dimensions of thechambers 42 are relatively small, there may be some difficulties addingthe beads into the chambers 42 while avoiding sample loss (e.g., beadspilling over the top of the flowcell 14).

FIGS. 16A and 16B provide a preferred embodiment of a deposition tool 70configured to facilitate the addition of beads to the reaction chamber42. As shown, the deposition tool 70 can include a series of inlet andoutlet couplings 72, 73 and an alignment pocket 71. The couplings 72, 73can include 0-rings (not shown) sized and configured to provide aneffective seal with a sample delivery instrument (e.g., a pipette, notshown). In use, as shown in FIG. 16B, the deposition tool 70 can couplewith the low-volume flowcell 14 in order to form a deposition assembly72 wherein the inlet and outlet couplings 72, 73, as well as thealignment pocket 71, are aligned with the inlet and outlet ports 44, 46of the underlying flowcell 14. The bead-delivery instrument can thensealably engage the inlet and outlet couplings 72, 73 and effectivelydeliver sample to the reaction chambers 42. The deposition tool 70 canbe formed of various materials. For example, the tool 70 can be formedof clear polycarbonate plastic plate.

Those skilled in the art will appreciate that various other embodimentsof a deposition tool are within the spirit and scope of the presentdisclosure. For example, in one embodiment, not shown, the tool can bebuilt onto a clamp to hold the flowcell in place with the beads areintroduced into the inlet/outlet couplings. All such embodiments arewithin the spirit and scope of the present disclosure.

In addition to facilitating sample introduction, in one embodiment, thedeposition tool 70 can be utilized during shipment and/or processing ofthe flowcells. That is, flowcells may be shipped as part of thedeposition assembly in order to provide added stability/security to theflowcell thereby preventing or reducing damage. Also, the flowcell canremain part of the deposition assembly during processing steps (e.g.,rotation and/or centrifugation of the flowcells during beadincorporation/binding to the flowcell substrate) prior to analysis.

In addition to the various embodiment of the low-volume sequencingapparatus 12 provided above, the presently disclosed system 10 furtherincludes various embodiments of a precision liquid delivery system 26 incommunication with the apparatus 12. That is, prior sequencing systemswaste large amounts of reagents due to extensive plumbing requirementswherein reagents must be transported through extensive tubing to thereaction chambers. Additionally, many reagent delivery systems involvedelivery lines, valves, etc., which suffer from a residual ‘dead volume’of reagents required for the actual transport process, but not actuallyutilized in the destination reaction chamber.

Minimizing reagent consumption in sequencing analysis is important forcost effectiveness. The reagent consumption can essentially be brokeninto two components including the actual reagent volume contained in thesequencing/reaction chamber and any reagent ‘overhead’ required in theprocess of transporting the reagent from its storage location to theactual point of use in the reaction chamber. As disclosed herein, theprecisions reagent delivery mechanism can reduce the system's ‘deadvolume’ to zero or near zero.

The currently disclosed system 10 includes a precision liquid reagentdelivery mechanism 26 configured to receive a pre-determined amount ofreagent from a reagent storage container, and further configured todeliver the entire pre-determined amount to the reaction chamber via theinlet port thus eliminating essentially all plumbing/tubingrequirements. Additionally, in one embodiment, this pre-determinedvolume can be substantially equal to the volume of the target reactionchamber. Thus, the delivery mechanisms can be configured to withdraw anexact amount of reagent from a storage container and then dispense thisentire or substantially this entire amount into the reaction chamber toachieve the desired effect (e.g., reaction). In other embodiment, thereagent delivery mechanism 26 can be configured to withdraw and retainan amount of reagent substantially equal to that amount required tointroduce reagent into each of the reaction chambers (or at least somesubset thereof). These improvements decrease analysis time, increaseaccuracy as a precise amount of reagent is being delivered to a desiredlocation, and substantially reduce waste thus lowering total cost.

FIG. 17 provides a preferred embodiment of the precision fluid/reagentdelivery mechanism 26. As shown, reagents can be transported fromvarious wells or containers 84 of an on-instrument reagent storagecontainer 28 to the flowcell 14 by means of an x-y-z robot 76, whichcarries or is configured in connection with, for example, a fluidicdispenser 78. In one embodiment, the fluidic dispenser 78 is a syringepump. At the storage container 28, the syringe pump 78, with apre-charge of air, aspirates just that amount of reagent required tofill the target reaction chamber 42 (or the plurality of reactionchambers) of the flowcell 14. The fluidic dispenser 78 can include anextension 80 having an internal chamber 82 sized and configured to houseand retain the reagent during transport from the reservoir 28 to thechamber 42. The robot 76 can reversibly couple the tip of the extension80 to an inlet port of the flowcell, making a sealing engagement. Theextension 80 can seal directly with the inlet port of the flowcell 14or, as shown, the extension can form a seal with a corresponding portionof a locking apparatus 86 overlying the flowcell 14. The entire requiredreagent volume can then be dispensed/pushed into the flowcell by the airpre-charge in the syringe, with little or no resulting dead volume. Inother embodiments, the syringe/extension 80 can retain enough reagent todispense an amount of the reagent into each of the reaction chambers ofthe flowcell or into each of the reaction chambers of multipleflowcells.

When it is time to empty the reaction chamber for the nextreagent/reaction, the syringe pump 78, charged with air or a washreagent, forces the reagent from the reaction chamber 42 thereby leavingthe chamber 42 in a state where it is occupied only by air or washliquid. Additional washes can then be performed as required, againleaving the chamber 42 in a state occupied only by air. Subsequentreagent deliveries and washes are repeated as outlined above. Becauseeach delivery is bounded by air, there is no liquid diffusion bordereffect that results in dilution.

Also disclosed herein is a mechanism for converting a relativelyhigh-volume sequencing system to a low-volume system. That is,low-volume applications of instruments for next generation sequencingsuch as for use with a sample volume of approximately 25 μl can bedesirable. Certain conventional systems have a significantly largersample volume of approximatly 400 μl or more and may not be able toefficently handle small volumes. As shown in FIG. 18, incorporation ofan injector port 95 into a fluidic handling system and flowcell canprovide a convenient mechanism to convert the system to be able tohandle small volumes. In various embodiments, this port 95 can beremotely located from the flowcell and enable the sample to move whilechemistry is being performed.

The port 95 can be configured to act like a valve that allows theexisting robot tip or other pipetting tip to be plugged into it toshorten the fluidic path and avoid moving the sample through the syringeand through the existing long tubing. The port can also be configuredwith a lid or top 96 that has at least one tubing 98 connected to it.This tubing 98 can be attached to a valve block so it can configured forself washing of the port 95 and the sample slide, and can also pump bulkreagents through the slide. The port 95 can further include a plurlaityof sealing mechansims 100, 102, 104 (e.g., O-rings) configured toeffectively seal the connection between the input tubing 96 and theoutput tubing 106 running to the flowcell. Desirable features of suchdesigns allow the system to handle smaller volumes with little or nodead volumes and fluid losses. Additionally, the port 95 can be used onexisting products that are required to pipette small volumes and/or loada sample.

FIG. 19 provides another view of the precision reagent deliverymechanism 26 relative to the flowcell 14 and low-volume sequencingapparatus 12. As shown, the x-y-z robot 76 can be positioned incommunication with the reagent storage containers 28 and the flowcell14. The reagent storage container 28, which can be provided as a kit,can include any number and/or type of reagents capable of being utilizedfor the desired analysis. Those skilled in the art will appreciate thata wide-range of such containters including a wide-range of differentreagenets, buffers, etc. are within the spirit and scope of the presentdislcosure.

In various embodiments, the low volume flowcell and robotically assistedreagent delivery system can be configured for use in next generationsequencing systems such as that provided by Life Technologies (e.g.,SOLiD). For example, Assignee's PCT Application Publication No. WO2006/084132, entitled “Reagents, Methods, And Libraries for Bead-BasedSequencing,” the entirety of each of these applications beingincorporated herein by reference thereto, describe a system for nucleicacid sequence anaysis using a flowcell imaging apparatus which maybenefit from the presently disclosed low-volume flowcell designsdisclosed herein, as well as from the automation capabilities providedby the robotically assisted reagent delivery apparatus.

Referring again to FIG. 1, the system can include a controller 30configured to control various aspects of the system. For example, thecontroller 30 can control manipulation of the flowcell(s) and theprocessing stage relative to the optics while also maintaining a desiredtemperature profile within the reaction chambers. In additional, thecontroller 10 can be configured to control the precision reagentdelivery mechanism such that precise amounts of specific reagents areintroduced to reaction chamber(s) at the precise time point duringsample analysis.

The controller can include various embodiments of a computer systemconfigured to control the flowcell(s), processing stage, temperatureprofile, precision reagent delivery mechanism, etc. For example, FIG. 20is a block diagram that illustrates a computer system 200, upon whichembodiments of the present teachings may be implemented. Computer system200 includes a bus 202 or other communication mechanism forcommunicating information, and a processor 204 coupled with bus 202 forprocessing information.

Computer system 200 also includes a memory 206, which can be a randomaccess memory (RAM) or other dynamic storage device, coupled to bus 202for issuing instructions to be executed by processor 204. Memory 206also may be used for storing temporary variables or other intermediateinformation during execution of instructions to be executed by processor204. Computer system 200 further includes a read only memory (ROM) 208or other static storage device coupled to bus 202 for storing staticinformation and instructions for processor 204. A storage device 210,such as a magnetic disk, optical disk, EPROM or the like is provided andcoupled to bus 202 for storing information and instructions.

Computer system 200 may be coupled via bus 202 to a display 212 (e.g.,the user interface 32), such as a cathode ray tube (CRT) or liquidcrystal display (LCD), for displaying information to a computer user. Aninput device 214, including alphanumeric and other keys, touchscreen,etc. may be coupled to bus 202 for communicating information and commandselections to processor 204. Another type of user input device is cursorcontrol 216, such as a mouse, a trackball or cursor direction keys forcommunicating direction information and command selections to processor204 and for controlling cursor movement on display 212. This inputdevice typically has two degrees of freedom in two axes, a first axis(i.e., x) and a second axis (i.e., y), that allows the device to specifypositions in a plane.

A computer system 200 can be implemented in connection with the presentteachings for purposes of executed predefined instructions, scripts, orreal-time operator issued commands. Consistent with certainimplementations of the present teachings, the computer system mayperform various operations associated with control, monitoring, and dataacquisition to thereby permit automated or semi-automatedfunctionalities. For example, the computer system 200 may be used toinvoke and execute desired workflows on the instrument, perform andevaluate instrument diagnostics and operational assessments for thevarious instrument components, obtain signals and information from theinstrument or components thereof, acquire sample data and processresults, and output information and data to the user. As will beappreciated by one of skill in the art, information and commands issuedand received by the computer system 200 may be in response to processor204 executing one or more sequences of one or more instructionscontained in memory 206. Such instructions may be read into memory 206from another computer-readable medium, such as storage device 210 ortransmitted across a network by another remote computer.

In one exemplary embodiment, execution of the sequences of instructionscontained in memory 206 may cause processor 204 to perform the desiredoperational functionalities including for example, control, monitoring,data acquisition, and data analysis processes. Alternatively hard-wiredcircuitry may be used in place of or in combination with softwareinstructions to implement the present teachings. Thus implementations ofthe present teachings are not limited to any specific combination ofhardware circuitry and software. Furthermore, computer system 200 may bea remotely located computer or part of a network of computers such as adistributed or cloud computing environment. Furthermore, otherelectronic devices such as PDAs, cellular phones, laptops or otherportable or detached devices may be interconnected with computer system200 as well as directly or indirectly to the instrument to provideselected functionalities as described above. In various embodiments,these other electronic devices may desirably be used to provide selectedfunctionalities such as monitoring the runtime operation of theinstrument, obtaining/downloading sample data, transmitting operationsinstructions, etc.

The term “computer-readable medium” as used herein refers to any mediathat participates in providing instructions to processor 204 forexecution. Such a medium may take many forms, including but not limitedto, non-volatile media, volatile media, and transmission media.Non-volatile media includes, for example, optical or magnetic disks,such as storage device 210. Volatile media includes dynamic memory, suchas memory 206. Transmission media includes coaxial cables, copper wire,and fiber optics, including the wires that comprise bus 202.

Common forms of computer-readable media include, for example, a floppydisk, a flexible disk, hard disk, magnetic tape, or any other magneticmedium, a CD-ROM, any other optical medium, punch cards, papertape, anyother physical medium with patterns of holes, a RAM, PROM, and EPROM, aFLASH-EPROM, any other memory chip or cartridge, or any other tangiblemedium from which a computer can read.

Various forms of computer readable media may be involved in carrying oneor more sequences of one or more instructions to processor 204 forexecution. For example, the instructions may initially be carried on themagnetic disk of a remote computer. The remote computer can load theinstructions into its dynamic memory and send the instructions over anetwork or telephone line. A network or modem interface local tocomputer system 200 can receive the data and convert the data to asignal or format of instructions recognized by the instrument. Theinstructions may optionally be stored on storage device 210 eitherbefore or after execution by processor 204.

In accordance with various embodiments, instructions configured to beexecuted by a processor to perform a method are stored on acomputer-readable medium. The computer-readable medium can be a devicethat stores digital information. For example, a computer-readable mediumincludes a compact disc read-only memory (CD-ROM) as is known in the artfor storing software. The computer-readable medium is accessed by aprocessor suitable for executing instructions configured to be executed.

FIG. 21 is schematic diagram of a system 400 for processing a sample, inaccordance with various embodiments. System 400 includes sample analysiscomponent 410 and processor 420. The sample analysis component 410 caninclude, but is not limited to including, hardware associated with fluidhandling, imaging 412, optics 414, and detector 416. In variousembodiments, the sample analysis component may comprise a nucleic acidsequencer 410 such as a next generation DNA sequencing (NGS) system.Nucleic acid sequencer 410 may be capable of interrogating a sample,produces reads from the sample indicative of the composition, andprovide the ability to assemble or analyze the data obtained from theinstrument.

Processor 420 is in communication with nucleic acid sequencer 410.Processor 420 can be, but is not limited to, a computer, microprocessor,or any device capable of sending and receiving control signals and datafrom nucleic acid sequencer 410 and processing data. Processor 420 maybe configured to perform a number of steps. Processor 420 may obtain theraw data or reads from sequencer 410. Processor 420 may further obtain areference sequence or genomic information used in further analysis andassembly of the data obtained from the sequencer. In variousembodiments, the reference sequence may be retrieved from a database,for example. The database can be a physical storage device with its ownprocessor (not shown) that is connected to processor 420 across anetwork, or it can be a physical storage device connected directly toprocessor 420, for example. Processor 420 may be configured to performselected analysis in addition to the operations/functionalitiesdescribed above.

In addition to the various devices, systems, and computer systemsprovided above, various methods for optimizing sample analysis are alsoprovided herein. For example, the methods include automating fluiddelivery to a low-volume flowcell thereby eliminating (or substantiallyreducing) reagent waste and allowing for system optimization. In oneembodiment, the method includes providing an automatic reagent deliverymechanism capable of moving between one or a plurality of reagent samplecontainers and the low-volume flowcell.

That is, in one embodiment, the method allows for the automated reagentdelivery mechanism to withdraw a precise amount of a specific reagentfrom a reagent sample container and deliver this pre-determined,specific amount of reagent to a desired channel of the low-volumeflowcell. The mechanism can be an x-, y-, z-configured roboticinstrument having, for example, a syringe pump associated therewith.Once the syringe pump of the mechanism withdraws the pre-determinedamount of reagent, the mechanism can reversibly couple with an inletport of the low-volume flowcell in order to place an internalcompartment of the delivery mechanism into fluid communication with thereaction chamber associated with the inlet port. As detailed above, theinlet port can be sized in configured in various manners in order toallow for an effecting coupling (e.g., formation of a seal) between themechanism and the inlet port.

In other embodiments, the delivery mechanism can be configured and/orprogrammed to perform many functions and/or to move between variousinlet ports. For example, the delivery mechanism can be configured tocouple/decouple a plurality of distinct inlet ports in order to dispensea pre-determined amount of a reagent to a plurality of distinctchannels. Thus, the mechanism can allow a pre-determined amount of thesame reagent to be delivered to a plurality of channels, or can allowfor distinct reagents to be delivered to different channels therebyallowing for distinct protocols to be performed in each (or at leasttwo) of the reaction channels.

One skilled in the art will appreciate further features and advantagesof the invention based on the above-described embodiments. Accordingly,the invention is not to be limited by what has been particularly shownand described, except as indicated by the appended claims. Allpublications and references cited herein are expressly incorporatedherein by reference in their entirety.

What is claimed is:
 1. A low-volume sequencing system, comprising: alow-volume flowcell having at least one reaction chamber of a definedvolume and at least one fluid transfer port formed therein; and anautomated reagent delivery mechanism having a fluid dispenser with aninternal compartment configured to retain a selected amount of reagent,the fluid dispenser having a distal portion configured to reversiblycouple with the inlet port thereby placing the internal compartment intofluid communication with the at least one reaction chamber, and furtherconfigured to dispense to the reaction chamber a volume substantiallyequal to the defined volume of the reaction chamber.
 2. The system ofclaim 1, wherein the delivery mechanism is configured to retain a volumeof reagent substantially equal to the volume of the reaction chamber. 3.The system of claim 1, further comprising a controller in communicationwith the delivery mechanism, and configured to couple or decouple thedelivery mechanism with the fluid transfer port, and further configuredto dispense reagent to the reaction chamber.
 4. The system of claim 1,wherein the defined volume of the reaction chamber is less than about100 μl.
 5. The system of claim 1, wherein the flowcell includes aplurality of reaction chambers, each reaction chamber having a discretevolume.
 6. The system of claim 5, wherein each reaction chamber extendsbetween at least two discrete fluid transfer ports.
 7. The system ofclaim 5, wherein the internal compartment of delivery mechanism isconfigured to retain a volume of a reagent substantially equal to atotal volume of the plurality of reaction chambers.
 8. The system ofclaim 1, wherein at least one fluid transfer port is incorporated into atop portion of the low-volume flowcell.
 9. The system of claim 1,wherein at least one fluid transfer port is incorporated into a bottomportion of the low-volume flowcell.
 10. The system of claim 1, whereinat least a portion of the flowcell surface is configured to bind asample.
 11. The system of claim 10, wherein the sample is apolynucleotide or the sample is a solid support configured to bind apolynucleotide or a polynucleotide.
 12. The system of claim 1, whereinan interior surface of the flowcell is configured to bind sample. 13.The system of claim 1, wherein the reagent delivery mechanism includes arobotic assembly having x-, y-, and z-functionality.
 14. The system ofclaim 1, wherein the low-volume flowcell includes a plurality ofdiscrete reaction chambers.
 15. The system of claim 1, furthercomprising a reagent storage container housing a plurality of reagents.16. A low-volume flowcell, comprising: a substrate having at least onereaction chamber of a defined volume extending between an inlet port andan outlet port, the inlet port being configured to reversibly couplewith an automated reagent delivery mechanism which is configured todeliver a pre-determined amount of reagent to the reaction chamber, thereaction chamber being sized and configured to minimize thepre-determined amount of reagent required to effect a desired result.17. The low-volume flowcell of claim 16, wherein the defined volume isbetween about 15 μl and about 35 μl.
 18. The low-volume flowcell ofclaim 16, wherein the defined volume is between about 10 μl and about 40μl.
 19. The low-volume flowcell of claim 16, wherein the defined volumeis about 25 μl.
 20. A method of analyzing a biomolecule, comprising:providing an automated reagent delivery mechanism configured to withdrawa pre-determined volume of a reagent from a reagent storage container;withdrawing a pre-determined amount of the reagent from the storagecontainer by the automated reagent delivery mechanism; coupling aportion of the automated reagent delivery mechanism to an inlet port ofa flowcell, the inlet port being in fluid communication with a reactionchamber having a defined volume; dispensing into the reagent chamber areagent volume substantially equal to the defined volume of the reagentchamber; and decoupling the automated reagent delivery mechanism fromthe inlet port.